Exploring Alkyl-O-Alkyl Ether Structures in Softwood Milled Wood Lignins

Recent studies have suggested that there are significant amounts of various alkyl ether (Alk-O-Alk; Alk = alkyl) moieties in a spruce native lignin preparation, milled wood lignin (SMWL). However, the comprehensive NMR assignment to these moieties has not been addressed yet. This study focused on investigating different types of Alk-O-Alk structures at the α- and γ-positions of the lignin side chain in an heteronuclear single-quantum coherence (HSQC) spectrum of SMWL using experimental NMR data of lignin and synthesized model compounds. Ambiguous structural features were predicted by computer simulation of 1H and 13C NMR spectra to complement the experimental NMR data. As a result, specific regions in the HSQC spectrum were attributed to different Alk-O-Alk moieties of Alk-O-Alk/β-O-4 and Alk-O-Alk/β-β′ structures. However, the differences between the specific regions were rather subtle; they were not well separated from each other and some major lignin moieties. Furthermore, SMWL contained a large variety of Alk-O-Alk moieties but in minute individual amounts, resulting in rather broad, superimposing resonances. Thus, evaluation did not allow assigning individual types of Alk-O-Alk moieties from the HSQC spectra; instead, they were quantified as total (α- and γ-linked) Alk-O-Alk based on the balance of structural units in the 13C NMR spectra. At last, potential formation mechanisms of various Alk-O-Alk ether structures in lignin biosynthesis, lignin aging, and during ball milling of wood were hypothesized and discussed.


■ INTRODUCTION
Lignin is a very complex heterogeneous aromatic polymer. Despite the tremendous number of studies, some elements of its chemical structure are still under discussion. For example, the current analytical methodology allows only the description of 80−85% of structural moieties in native lignin preparations, such as milled wood lignins (MWLs). 1−3 The structures of the remaining 15−20% of lignin units are not well understood. Based on the overall material balance and specific features in the 13 C NMR spectra along with literature data for comparison, they were tentatively assigned to various types of alkyl ether (Alk-O-Alk) moieties at the αand γ-positions of the side chain. 1−3 Alk-O-Alk moieties, that is, aliphatic ether structures, may play an important role in lignin branching�in agreement with our view that the macromolecular structure of lignin is a threedimensional network rather than a linear chain 1,2 �in addition to already identified structural units, such as etherified biphenyl (5-5′) and diaryl ether (4-O-5′) structures, which are among the main branching points in lignin. It was suggested that about 20−28% of monolignols in softwood lignins were involved in 5-5′ (major) and 4-O-5′ (minor) linkages based on CuO-permanganate oxidation (PO), 13 C NMR, and thioacidolysis-31 P NMR methods. 1,2,4,5 The analysis of the absolute molecular mass and the number of terminal units in spruce MWL (SMWL) showed that it is even more branched/ crosslinked than expected solely from the quantification of the known branching points at the aromatic rings. 2 Only half of the branching points were located there, whereas the other half was expected to be located in the side chains. They were assigned to various Alk-O-Alk structures at the αor γpositions of the side chain, with additional β-O-4 or β-β′ linkages (Alk-O-Alk/β-O-4, β-β ether structure). 2 Although the occurrence of Alk-O-Alk structures in native lignin is still not commonly accepted, their presence has been proposed and discussed earlier. 6−10 For the noncyclic Alk-O-Alk/β-O-4 ether structures, Leary 6,7 proposed that 17−21 benzyl noncyclic alkyl ether groups per 100 C9 units might occur in spruce Bjorkman lignin, based on Adler's studies. 8 Glasser et al. 9 suggested 25% of noncyclic α-O-γ′ ether linkages based on computer simulation of softwood lignin structure. Another piece of evidence was provided by Sakakibara's group, who isolated an α-O-γ′ ether dimer from lignin hydrogenolysis products. 10 Regarding the cyclic Alk-O-Alk/β-β′ ether structure, cyclic α-O-α′ and α-O-γ′ ether linkages were reported by Ralph and Lu,11 identified in syringyl lignin from palm, kenaf, and corn cell walls. Zhang and Gellerstedt 12 even found a guaiacyl analogue of cyclic α-O-α′ether with β-β′ linkages in an SMWL in relatively small amounts but no a γ-O-Alk ether analogue. Bicyclic epiresinol structures have previously been suggested for hardwood kraft lignin by Bruijnincx et al. 13 Alk-O-Alk moieties are often unstable toward wet chemistry methods used in lignin analysis, which easily transform into aliphatic alcohols. Therefore, they are difficult to detect upon degradative analysis of lignin. Nondegradative techniques, in particular NMR spectroscopic techniques, such as heteronuclear single-quantum coherence (HSQC), are especially valuable for their direct detection. Recent scrutiny of softwood MWL (including 2D NMR) showed some unassigned signals, 2,3 which so far had been disregarded. We suggested they belong to a variety of Alk-O-Alk ether structures in general. 1−3 However, their structural information is still ambiguous, and the exact formation during lignin biosynthesis is still unclear. Therefore, the present study is devoted to the elaboration of Alk-O-Alk ether moieties in SMWL ( Figure 1) using experimental data of lignins and relevant model compounds complemented by computer modeling. A selection of most important structures under discussion will have to be further corroborated by synthesis of specific model compounds and their NMR analysis in future studies. ■ EXPERIMENTAL SECTION General. All chemicals and solvents were purchased from Sigma-Aldrich and used without further purification, except dioxane which was distilled over sodium hydroxide before each use. The information on the selected model compounds is described in the Supporting Information. Computationally unsophisticated simulation of 1 H and 13 C NMR spectra was performed with the ChemDraw Professional v19.0 package. 14−18 Isolation of MWL. Spruce (Picea abies) wood meal (60 mesh pass) was preextracted with ethanol/toluene, 1:2 (v/v), in a Soxhlet apparatus to remove lipophilic extractives. The MWL preparation was isolated from the preextracted wood meal and purified as described earlier. 3,19 The yield of the purified SMWL was about 25% per Klason lignin content in wood.
HSQC NMR Experiment. The MWL (80 mg) was fully dissolved in 0.6 ml of DMSO-d 6 . A high-resolution HSQC spectrum was acquired with a Bruker AVNEO 600 MHz spectrometer equipped with a 5 mm He-cooled TCI gradient cryoprobe using a Bruker pulse program "hsqcetgpsisp.2" with maximum sensitivity enhancement. 1024 data points were acquired at 298 K, from 11 to 0 ppm in F2 ( 1 H), with an acquisition time of 77.8 ms, and from 215 to 0 ppm in F1 ( 13 C) with 256 increments, 36 scans, and a 2.0 s interscan delay. The heteronuclear coupling constant value was set at 145 Hz. Processing the final matrix to 2 K by 1 K data points was performed by QSINE window functions in both F2 and F1. The spectral processing was carried out with Bruker's Topspin 4.0 (Windows) software. The central peak of the residual solvent (δ H 2.49, δ C 39.5 ppm) was used for calibration. All known correlation peaks were assigned based on earlier reports. 2,3,20−26 Figure 2. Partial short-range 1 H− 13 C HSQC NMR spectrum (oxygenated aliphatic region) of SMWL, in DMSO-d 6 . The carbohydrate content was calculated by integration shown in Figure S1. reported the carbohydrate content of about 0.7%. Therefore, the influence of carbohydrates on the analysis of lignin structure was very minor for this sample.
Two approaches were followed to explore the presence of proposed Alk-O-Alk ether structures in SMWL: model compounds with Alk-O-Alk ether moieties were synthesized (Figure 3), and some of their NMR data were reviewed in light of previous studies (Table 1). 11,28−33 To address additional structural features, this was complemented by simulated Alk-O-Alk models and their spectra ( Figure 5), which were plotted against the experimental HSQC spectrum of SMWL to assist with possible assignments.
Structural Identification Based on Experimental NMR Data. As shown in Figure 3 and Table 1 The NMR data of models 1, 4, and 8 were recorded in DMSO-d 6 , which were the same as those recorded in the solvent for the HSQC spectrum of SMWL. The direct spectral comparison is shown in Figure 4a,b. The other model compounds were analyzed in CDCl 3 , and the chemical shift difference between SMWL and model compounds caused by the solvent effect had to be considered in this study. According to the NMR database of lignin compounds, 20 the variation between DMSO-d 6 and CDCl 3 was generally 0.1 ppm for 1 H and 2 ppm for 13 C (Δδ H = ∼0.1 ppm, Δδ C = ∼2 ppm) or less. As a rule of thumb, CDCl 3 gave a higher δ C (downfield shift by up to 2 ppm) and a higher δ H (downfield shift by up to 0.1 ppm) than DMSO-d 6 , which need to be considered when comparing the NMR data with SMWL measured in DMSO-d 6 . As a consequence, the central points of the cross-peaks in spectra measured in CDCl 3 should shift toward the upper right quadrant in the circled regions when making the transition to DMSO-d 6 , as indicated by the colored squares in Figure 4c,d. In addition, NMR data for LCC model compounds in Table  S3 and S. Ralph's database 20 indicated that acetylation at the γposition affected the chemical shifts of γand β-CH but not that at the α-position. Therefore, the NMR data of the α-alkyl positions in the γ-acetylated model compounds 3, 6, and 7 can be directly used and compared with those of nonacetylated lignin.
When the chemical shift values of α-O-Alk (α-O-α′ and α-O-γ′)/β-O-4 ether-type model compounds collected in DMSO-d 6 (from Table 1) were superimposed with those of the SMWL spectrum (Figure 4a), it was evident that the H α − C α and H β −C β correlations from models 1 and 4 matched well, suggesting that the H−C correlation cluster 1 (δ H /δ C , 4.1− 4.9/77−83 ppm) in the SMWL spectrum comprises both    Table S3, one can say that there was no difference in the chemical shift of the side-chain CH between the syringyl and guaiacyl β-β′ model compounds. The NMR data of syringyl model compounds 3 and 7 can thus be directly used in lieu of the guaiacyl analogue. As shown in Figure 4a, the H α −C α correlations from α-O-Alk/β-β′ether compounds 3 and 7 were close to those of typical DBDO and resinol structures. As seen in Figure 4d Structural Identification with Simulated NMR Data. Computational models can help to assign Alk-O-Alk ether structures and to extensively explore structure−shift correlations, especially in cases of synthetically hard to access model compounds or when minor structural differences are to be studied. Of course, there is some limitation caused by computational error limits and an uncertainty with regard to consideration of solvent effects, yet the method is still very helpful and widely applied in structural identification. The model compounds VG, GG, and GH in Table S3 from previous studies 20, 34 was employed to evaluate the quality of  the computational estimation of the NMR data. The difference between experimental and simulated NMR shifts, as shown in Table S3, was significant both for the 1 H and 13 15) showed that the effect of γ′-alkyl etherification of another β-O-4 unit (in structures II or III) was larger than that of α′-alkyl (in structure I), with Δδ C = ∼8−14 ppm for γ′-Alk being larger than Δδ C = ∼6−11 for α′-Alk. This was consistent with a tendency observed for the synthesized model compounds (VG vs models 1E, 2, 6, and 9E in Table S4). Thus, in Figure 6, the spectral region 1 and the spectral region 2, indicated by circles, were specifically attributed to α-CH of
Of particular interest was the change of the δ H /δ C value of β-CH for VG after alkyl etherification (Table 2). Etherification at the α-position or γ-position would cause a downfield shift in the δ H of β-CH (Δδ H = ∼+0.2 ppm) and an upfield shift in δ C (Δδ C = ∼−2 ppm). This was also in agreement with results from experimental NMR data (Table S4). For example, alkyl etherification at the α-CH of VG with an α-alkyl moiety from another side chain would cause a downfield shift in δ H (Δδ H = ∼+0.4 ppm) and an upfield shift in δ C (Δδ C = ∼−6 ppm) for     (Table 3).
The chemical shifts of β-CH in synthesized model compounds 3, 7, and 10 could not be evaluated directly for the corresponding nonacetylated moieties because acetylation strongly affects the resonance of β-CH but through simulation studies. There was an upshift in δ H /δ C of β-CH in these models relative to the bicyclic β-β′ resinol structure (Table 3).
Based on this trend, it was concluded that the region for β-CH of α-O-Alk/β-β′ and γ-O-Alk/β-β′ ether structures should appear around 48−51/2.0−2.2 ppm. However, no significant resonance was observed in this area of the spectrum ( Figure  6). This might be due to the much lower response factor of β-CH as compared to that of γ-CH 2 in the same structures (observed in the lower field, around 65−73/3.5−4.0 ppm) and implied a high structural variety of these moieties with a very minor amount of each specific structure.
In summary, our studies did not only support the earlier tentative assignment of the regions for Alk-O-Alk ethers involved with β-O-4 structures 2 but also provided information on the chemical shift of various types of moieties involved in β-β′ structures. These substructures were not completely separated in HSQC spectra to be reliably quantified individually. Moreover, some overlapped with the canonical lignin moieties, such as β-O-4/α-OH, pinoresinol, SD, and DBDO. Therefore, Alk-O-Alk moieties can only be quantified as sum (α-and γ-ethers) based on the material balance in 13 C NMR. 2,3 Possible Formation Pathways of Alk-O-Alk Ether Structures in SMWL. Radical Coupling Theory. According to the generally accepted radical coupling theory 35,36 Figure  3) by the reaction between a QM and dihydroconiferyl alcohol. 29 Herein, we speculate that QMs generated via the β-O-4 type radical coupling reaction may react with α-OH or γ-OH from another lignin fragment to give rise to a series of Alk-O-Alk/β-O-4 ether structures. However, we could not suggest an appropriate analogous mechanism for the formation of γ-O-γ′/β-O-4 ether structures during lignin biosynthesis so far.
Novel types of β-β′ structures with one opened ring, identified in syringyl lignin from palm, kenaf, and corn cell walls by Ralph and Lu,11 also have Alk-O-Alk ether moieties. Unlike the proposed Alk-O-Alk/β-O-4 ether structures, the resinol β-β′structure was produced by internal trapping between the β-β′-bonded QM (QM2 in Scheme 1a) and its two γ-OH. However, if the γ-OH of sinapyl alcohol was acylated, the β-β′ homo-coupling of γ-acylated sinapyl alcohol formed an intermediate bis(quinone methide) (QM4 in Scheme 1b). Since γ-acetylation prevented the internal attack of the γ-OH on QM4, it re-aromatized by water addition, see the typical water addition to QM1. The resultant α-OH intermolecularly added to another QM to form a cyclic α-O-α′/β-β′ether structure with one opened ring. 11 The β-β′crosscoupling of a γ-acylated monolignol and a typical monolignol produced QM5 (Scheme 1b). There was an internal γ′-OH capable of trapping one QM moiety, forming an α-O-γ′ ether, while the other QM re-aromatized by water addition to form an α-OH, producing an α-O-γ′/β-β′ether structure. 11 Accordingly, the guaiacyl analogue of Alk-O-Alk/β-β′ ether linkages is proposed to occur similarly to the above formation of the syringyl analogue by the radical coupling mechanism (Scheme 1b).
Lignin Aging. In addition to the widely accepted dehydrogenation radical coupling mechanism for lignin biosynthesis, there was another proposal that a high number of Alk-O-Alk ether linkages could have been formed during aging of the lignin in plant tissues over the long growing periods. Leary 6 proposed that there was a potential for QMs to be transiently re-formed throughout the lifetime of the lignin polymer in the plant cell wall based on lignin model compound studies. 37,38 The reversibility of QM generation was well proven and widely used in biotechniques, such as DNA modification and drug release. 39,40 This process, therefore, would allow the addition reaction between the regenerated QMs from unstable p-hydroxy benzyl alcohol-type structures and lignin aliphatic alcohol nearby, resulting in a considerable amount of structurally variable Alk-O-Alk ether linkages over time (Scheme 2b). It seemed reasonable that the α-O-Alk ethers (see models 11 and 14) could also be produced during aging of lignin in plants over time.
Ball Milling. In addition to the possibility of the formation of Alk-O-Alk ether structures during lignin biosynthesis and aging, we also proposed the possibility of their production during ball milling (Scheme 3). According to previous model studies on the mechanochemistry of lignin, 41,42 structural change of lignin caused by milling proceeded via a radical cleavage of β-O-4 linkages. It is reasonable to assume that recombination and follow-up chemistry of the produced βradicals can also result in the formation of Alk-O-Alk ether structures during milling. More specifically, the propenyl alcohol-type structures transformed from the β-radicals could generate αor γ-O-Alk ether structures through a nucleophilic substitution reaction (Scheme 3).

■ SUMMARY
The NMR chemical shifts of lignin moieties of Alk-O-Alk ether types were dependent on the substituent type (α-ether or γether) and the type of substituent at the β-position (β-O-4 or β-β′ type). The present study allowed distinguishing between six different types of Alk-O-Alk moieties, specifically those of α-O-α, α-O-γ, and γ-O-γ types with β-O-4 and β-β′ substituents. However, the differences between the specific NMR shift regions were rather subtle, and they were not well separated from each other and from other major lignin moieties. Furthermore, SMWL contained a very high variety of Alk-O-Alk moieties in very minor amounts resulting in superimposed, broad signals. However, although not individually identifiable, the Alk-O-Alk moieties of different types were assigned to different spectral regions in the HSQC spectra and were quantified as the sum parameter (total αand γ-ethers) based on the material balance in 13 C NMR spectra. Plausible mechanisms of the formation of various Alk-O-Alk ether structures in lignin biosynthesis, lignin aging, and during ball milling of wood were proposed. ■ ASSOCIATED CONTENT
Carbohydrate content in SMWL, calculated NMR shift data for side chains of Alk-O-Alk/β-O-4 ether model compounds, calculated NMR shift data for side chains of β-β′ and Alk-O-Alk/β-β′ ether model compounds, experimental NMR shift data for the side chain of different reference model compounds, and structural information on synthesized and computational model compounds used in this study (PDF)